5-Hydroxymethylcytosine (5hmC) is a newly identified base modification in mammalian genomic DNA. TET proteins, a group of iron(II)/?KG-dependent dioxygenases, have been shown to utilize dioxygen to oxidize 5-methylcytosine (5mC) to 5hmC, and to further form 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in the mammalian genome. Both 5fC and 5caC can be recognized and excised by human thymine DNA glycosylase (TDG), followed by base excision repair (BER) to replace the modified cytosine with a normal cytosine in an active demethylation process. The 5mC oxidation derivatives of 5hmC, 5fC, and 5caC may also be passively diluted to the unmethylated stage. In certain tissues or cells, 5hmC can accumulate to relatively high levels, whereas both 5fC and 5caC exist in much lower levels in most cells consistent with their constant removal by TDG through BER. The TET-mediated 5mC oxidation has been shown to play vital functional roles in mammalian early development events such as epigenetic reprogramming following fertilization and differentiations of various mammalian progenitor cells. However, studies of these systems have been significantly hampered due to lack of effective technologies that can map the presence and locations of oxidized 5mC derivatives with limited genomic samples; in many mammalian early development events only hundreds to thousands of cells can be isolated for functional characterizations. Building on our successes in inventing enabling technologies that label and sequence 5hmC and 5fC, we propose several innovative approaches that can robustly map the locations of these cytosine modifications genome-wide with as few as 1,000 cells or less. We further propose to study demethylation following fertilization to validate the new methods we plan to develop. The proposed research will provide urgently needed tools for the PI's group and the broader scientific community to study a range of research questions that should reveal new fundamental knowledge of epigenetics, development, and various human diseases.